M.I. Verkhovsky

Flash-induced electrogenic events in the photosynthetic reaction center and bc1 complexes of Rhodobacter sphaeroides chromatophores

Electrogenic events in Rb. Sphaeroides chromatophores have been studied (i) electrometrically in the chromatophore/phospholipid-impregnated collodion film system and (ii) spectrophotometrically by measuring the electrochromic spectral shift of carotenoids. Under the conditions when the bc 1 complex and ubiquinone pool were oxidized at pH 7.5, the second flashwas shown to give rise to at least two additional electrogenic phases of τ values approx. 0.2 and approx. 20 ms, which were not induced by the first flash. The fast phase was resistant to the inhibitors of the bc 1 complex, antimycin A and myxothiazol. It seems to be due to the protonation of reduced Q B in the RC complex. The slow phase was partly inhibited by antimycin A and completely by subsequent addition of myxothiazol. The antimycin-sensitive constituent of the slow phase was τ ≈ 40 ms and its rise was non-exponential. The antimycin-insensitive, myxothiazol-sensitive constituent was τ ≈ 7 ms. A comparison of (i) the kinetics of cytochrome b h redox conversion induced by the first and second flashes and (ii) the electrogenic reactions sensitive to the Q-cycle inhibitors suggests that the myxothiazol-sensitive electrogenic phase is associated with the reduction of cytochrome b h ( b -561). The antimycin-sensitive electrogenic phase apparently results from the protonation of reduced Q in the quinone-reducing center of the bct complex. Reduction of ubiquinone to ubisemiquinone by b h seems to be electrically silent, since there is no electrogenic phase to follow the kinetics of this process. Myxothiazol addedin the absence of antimycin A induced a negative electrogenic phase with an opposite polarity (τ ≈ 2.5 ms) after the second flash. This phase, completely abolished by the addition of antimycin A, is assumed to be due to the electrogenic deprotonation of the RC-reduced QH 2 which combines with center C in the bc 1 complex. The data obtained by the electrometric and spectrophotometric methods appear to be very similar, though the electrometric method is more sensitive because of the much higher signal-to-noise ratio.

Electrogenesis associated with proton transfer in the reaction center protein of the purple bacterium Rhodobacter sphaeroides

Electrogenic events in the photosynthetic reaction center complex (RC), accompanying single‐ and two‐electron reduction of the secondar quinone acceptor Qb, were investigated. In the presence of inhibitors of electron transfer via the bc1‐complex, the kinetics of formation of the transmembrane electric potential difference induced by two successive light flashes exhibit a few phases. Besides the fast phase A which is due to the charge separation between the bacteriochlorophyll dimer P and primary quinone acceptor qa, two slower atrazine‐sensitive phases, BI and BII, were observed. Phase BI is suggested to be due to proton transfer between the amino acid residues of the reaction center protein, and phase BII due to proton uptake during the second flash‐induced formation of ubiquinol. A possible model of electrogenesis in the acceptor moiety of the RC is discussed.

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Abstract Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q ⨪ B ) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential ( E h ) to 200–300 mV is the result of increasing the lifetime of Q ⨪ B , as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q ⨪ B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q ⨪ B , the flash-induced redox changes of Q ⨪ B were observed at low E h values unless E h reached a value at which Q B underwent reduction at equilibrium to form Q B H 2 . The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q B H 2 ) of the secondary acceptor.

Partial reversion of the electrogenic reaction in the ubiquinol: Cytochrome c2-oxidoreductase of Rhodobacter sphaeroides chromatophores under neutral and alkaline conditions

The interaction of the photosynthetic reaction center (RC)‐generated ubiquinol with the ubiquinone‐reducing center C of ubiquinol:cytochrome c 2‐oxidoreductase (bc 1 ‐complex) has been studied electrometrically in Rhodobacter sphaeroides chromatophores. The addition of myxothiazol inhibited the ubiquinol‐oxidizing center Z, suppressing the phases of membrane potential generation by the bc 1,‐complex, but at the same time induced an electrogenic phase of opposite polarity, sensitive to antimycin A, the inhibitor of center C. The rise time of this reverse phase varied from 3 ms at pH 6.0 to 1 ms at pH 9.5. At pH > 9.5 the reverse phase was limited by the rate of ubiquinol formation in RC. The magnitude of the reverse phase was constant within the pH range 7.5–10.0. It is assumed that the reverse phase is due to the electrogenic deprotonation reaction which takes place after the binding of the RC‐generated ubiquinol to center C.

Transfer of ubiquinol from the reaction center to the bc 1 complex in Rhodobacter sphaeroides chromatophores under oxidizing conditions

The mechanism of interaction between the photosynthetic reaction center (RC) and bc 1 complex has been investigated in chromatophores of Rhodobacter sphaeroides. The kinetics of cytochrome b h reduction and formation of the transmembrane electric potential were measured at high E h, a condition where ubiquinol is formed in the RC only on the second light flash. In the presence of antimycin A, the kinetics of cytochrome b h reduction have been shown to be sensitive neither to the amount of ubiquinol produced nor to the number of active bc 1 complexes. It is concluded that the reaction between the ubiquinol produced on the second flash and the bc 1 complex is monomolecular. To explain the monomolecular pattern of this reaction under oxidizing conditions (the present work) and the previously described bimolecular pattern under reducing conditions [(1983) Biochim. Biophys. Acta 723, 202–218], it is proposed that (i) quinone exchange between the RC and bc 1 complex occurs via a local quinone pool and (ii) the rate of exchange between the quinone pools is very much slower than cytochrome b h reduction.

Effect of pH and surface potential on the rate of electric potential generation due to proton uptake by secondary quinone acceptor of reaction centers in Rhodobacter sphaeroides chromatophores

Abstract An electrometric method was used to investigate the effect of pH and ionic strength on the second flash-induced formation of the transmembrane electric potential difference (Δψ) arising from proton uptake by the quinone complex of the photosynthetic reaction center (RC) in chromatophores of non-sulphur purple bacterium Rhodobacter sphaeroides (wild-type). The characteristic time of this Δψ phase is approx. 70 μs at pH 7.5 in the presence of 20 mM KCI. Increases in both pH and salt concentration were found to slow the time of generation of the electric potential due to quinol formation (QBH2) after the second flash by more than 10-times. At higher pH, the effects of salts were more pronounced. The pH dependence of the Q2−B protonation rate is explained by the change in the surface proton concentration near the RC protein. The density of surface charges calculated on the basis of Gouy-Chapman theory is approx. 0.002 e/A2 (pH 7) or approx. 10 negative charges per RC protein from the cytoplasmic side of the membrane, in good agreement with previous estimates for chromatophores. The sensitivity of the rate constant of the second flash-induced RC protonation to the salt concentration probably resolves the contradiction among results reported by different laboratories (Wraight, C.A. (1979) Biochim. Biophys. Acta 548, 309-327; Kleinfeld, D., Okamura, M.Y. and Feher, G. (1985) Biochim. Biophys. Acta 809, 291-310) for the pH dependence of the rate constant of the reaction Q−AQ−B → QAQBH2.

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus.

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone‐10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash‐induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+Q− A) formation. Since the C aurantiacus RC lacks H‐subunit, this subunit was not an obligatory component for electrogenic QB protonation.

Properties of photosynthetic reaction centers isolated from chromatophores of Chromatium minutissimum

Abstract Functional characterisitcs of photosynthetic reaction centers from the sulphur purple bacterium Chromatium minutissimum were investigated. In the absence of the secondary quinone acceptor (Q B ), rereduction of P + following a flash occurs by recombination with the reduced primary quinone (Q A ) in approx. 40 ms, independent of pH within the range 6–10. The function of the secondary quinone acceptor is reconstituted by adding ubiquinone (Q−6), as indicated by the pronounced slowing of P + dark recovery and by flash-induced binary oscillations of semiquinone Q B formation. The time of the Q A − Q B → Q A Q B − and Q A − Q B − → Q A Q B 2− transitions is approx. 20 μs at pH 6.3. The spectrum of the reduced primary (Q A − ) and secondary (Q B − ) acceptor suggests that the primary quinone acceptor is menaquinone and secondary quinone acceptor is ubiquinone. It is found that the association constant for the secondary quinone acceptor in the RC protein, measured through the ratio of slow and fast components of pigment dark relaxation, is smaller in alkaline conditions than in acidic ones. The kinetics of flash-induced oxidation of high-potential heme, measured at 556 and 420 nm, were well approximated by a single exponential with τ ≅ 1.5 μ s. Analysis of the kinetics of cytochrome dark reduction by the reduced quinone acceptors (via pigment P) permits us to estimate the equilibrium constant of electron transfer between high-potential heme and P as 130, indicating E m ≅ 360 mV for C + /C couple. The equilibrium constant of electron transfer between Q A − Q B and Q A Q B − states of RC, estimated from kinetics of dark recovery of photooxidized pigment, is characterized by strong pH-dependence and is ≅ 22 at pH 10 and more than 300 at pH 6 with p K ≅ 7.6. The P + Q B − dark relaxation rate in Chromatium is pH independent at low pH, but slows down drastically in Rs. rubrum and Rb. sphaeroides (both contain ubiquinone as Q A and Q B ). These contrasting observations lead us to suggest that at low pH, the slow dark relaxation of pigment in Chromatium is determined by direct electron transfer from Q B (not through Q A ).

The Electrogenic Event Associated with the Reduction of the Secondary Quinone Acceptor in Rhodobacter Sphaeroides Reaction Centers

An electrometric method was used to investigate flash-induced electrogenic stages in proteoliposomes containing photosynthetic RCs of Rhodobacter sphaeroides. Besides the very fast electrogenic step associated with primary dipole formation, an additional electrogenic stage with a rise-time of 0.25 ms (pH 7.5) appeared to be induced by even numbered flashes. The maximal amplitude of this stage contributes ~0.3 to the fast phase, associated with the charge separation between P870 and QA. The similarity of the rise-time of this phase, the time of the disproportioning reaction of semiquinones QA and QB and the rate of proton uptake by RCs, its appearance only after even-numbered flashes, the sensitivity to o-phenanthroline as well as the increase of its rise-time with pH indicate that the additional electrogenic phase arises from the reaction: n n